This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mass spectrometry, the core technology in the field of proteomics, promises to enable scientists to identify and quantify the entire complement of proteins in a complex biological sample. CMass spectrometry, the core technology in the field of proteomics, promises to enable scientists to identify and quantify the entire complement of proteins in a complex biological sample. Currently, the primary bottleneck in this type of experiment is computational. Existing algorithms for interpreting mass spectra are slow and fail to identify a large proportion of the given spectra. We describe a database search program called Crux that reimplements and extends the widely used database search program SEQUEST. For speed, Crux uses a peptide indexing scheme to rapidly retrieve candidate peptides for a given spectrum. For each peptide in the target database, Crux generates shuffled decoy peptides on the fly, providing a good null model and, hence, accurate false discovery rate estimates. Crux also implements two recently described post-processing methods: a p-value calculation based upon fitting a Weibull distribution to the observed scores, and a semi-supervised method that learns to discriminate between target and decoy matches. Both methods significantly improve the overall rate of peptide identification. Crux is implemented in C and is distributed with source code freely to non-commercial users.